Research ArticleINNATE IMMUNITY

RELMα-expressing macrophages protect against fatal lung damage and reduce parasite burden during helminth infection

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Science Immunology  24 May 2019:
Vol. 4, Issue 35, eaau3814
DOI: 10.1126/sciimmunol.aau3814
  • Fig. 1 Tracing of RELMα expression in MΦs using RetnlaCre_R26tdTomato mice (see also fig. S6).

    (A) BMDMs were generated from RetnlaCre_R26tdTomato mice and stimulated with recombinant murine IL-4 (20 ng/ml) in vitro. RELMα_tdTomato indicates the RetnlaCre-induced expression of the tdTomato fluorescent reporter from the Rosa26 locus and was analyzed by flow cytometry at the indicated hours (h) after stimulation. (B) Flow cytometry dot plots show the frequencies of RELMα_tdTomato+ MΦs and other cell types that had expressed RetnlaCre at some stage during their development. Parental gates are given in fig. S6. (C) Bar diagram shows the frequencies (mean + SD) of RELMα_tdTomato+ expressors among total cells of the respective type, isolated from at least five mice.

  • Fig. 2 Expression of RELMα_tdTomato in tissues of naïve RetnlaCre_R26tdTomato mice (see also fig. S4).

    Indicated tissues isolated from naïve RetnlaCre_R26tdTomato mice were stained with BODIPY 493/503 dye for adipocytes, anti-CD68 for tissue MΦs, anti–SP-C for surfactant protein C-positive type 2 pneumocytes, anti–Pecam-1 for endothelial cells of blood vessels, and 4′,6-diamidino-2-phenylindole for nuclei. RELMα_tdTomato indicates endogenous RetnlaCre-induced expression of the tdTomato fluorescent reporter from the Rosa26 locus. Images on the far right show overlay of the respective channels. Scale bars, 50 μm.

  • Fig. 3 RELMα expression in alveolar versus peritoneal MΦs.

    (A) Immunofluorescence of fixed lung tissue (top) from RetnlaCre_R26tdTomato mice shows strong endogenous RELMα_tdTomato signal in the airways and lung parenchyma (red), but not in lung MΦs stained with anti-CD11c (green) and anti-CD68 (violet), as seen on the overlay image of the respective channels on the far right (merge). Bottom shows immunofluorescence of unfixed BAL cells, revealing bright RELMα_tdTomato+ Alv-MΦs stained with anti-CD11c (green) and anti–Siglec-F (violet). Scale bars, 50 μm. (B) The upper dot plots show gating for Alv-MΦs in Ly5.1 B6 → Ly5.2 RetnlaCre_R26tdTomato BM chimeras. Similar gating was applied for Ly5.2 RetnlaCre_R26tdTomato → Ly5.1 B6 chimeras. The lower dot plots show percentages of RELMα_tdTomato+ Alv-MΦs gated as above and isolated from the following mice (from left to right): RetnlaCre_R26tdTomato mouse lacking the RetnlaCre transgene as a negative control (R26tdTom), RetnlaCre_R26tdTomato mouse carrying the RetnlaCre transgene as a positive control, Ly5.1 B6 → Ly5.2 RetnlaCre_R26tdTomato chimeras, and Ly5.2 RetnlaCre_R26tdTomato →Ly5.1 B6 chimeras. Results are representative of three to five individual mice per group (see also fig. S7). (C) RELMα_tdTomato Alv-MΦs were sorted from BAL of RetnlaCre_R26tdTomato mice, labeled with CellTrace, and cultured for 48 hours in vitro with or without IL-4. Numbers indicate the frequency of RELMα_tdTomato+ cells among total Alv-MΦs. The lower histograms show dilution of the CellTrace proliferation dye after 48 hours of culture in the presence or absence of IL-4. (D) Relative mRNA expression of the indicated genes normalized to Hprt mRNA in sorted RELMα_tdTomato+ (black bars) and RELMα_tdTomato (white bars) MΦs from the peritoneum (pMΦs, top) or BAL fluid (Alv-MΦs, bottom). Data are obtained from >10 mice for isolating pMΦs and >30 mice for isolating Alv-MΦs from BAL of naïve RetnlaCre_R26tdTomato mice.

  • Fig. 4 STAT6 regulates expression of RELMα in a cell-specific manner.

    (A) Dot plots show percentages of RELMα_tdTomato+ cells among neutrophils (Ly6G+, Ly6C+), blood eosinophils (Siglec-F+, SSC-Hhi), pMΦs (CD11b+, F4/80+), and BAL Alv-MΦs (Siglec-F+, CD11c+) isolated from naïve RetnlaCre_R26tdTomato mice generated on a C57BL/6 (WT), STAT6ko, or IL-4/IL-13ko (4-13ko) background. (B) Bar diagram summarizes data in (A). Bars show the frequencies (mean + SD) of five individual mice per group and are representative of three independent experiments. **P < 0.01 by two-tailed Student’s t test. (C) Histograms show percentages of intracellular RELMα protein-expressing pMΦs [pregated as in (A) among cells positive for the RELMα_tdTomato reporter], isolated from WT and STAT6ko RetnlaCre_R26tdTomato mice. Data are representative of at least three to four mice per group. (D) Bar diagrams show concentrations of RELMα protein in serum, peritoneal, and BAL fluids harvested from WT and STAT6ko RetnlaCre_R26tdTomato mice and analyzed by enzyme-linked immunosorbent assay (ELISA). Data are pooled from at least three to four mice per group. (E) RELMα_tdTomato monocytes were sorted from BM of Ly5.2 RetnlaCre_R26tdTomato mice, labeled with CellTrace, and injected intraperitoneally into Ly5.1 B6 recipients (WT → WT), or from STAT6ko RetnlaCre_R26tdTomato mice into recipients lacking STAT6 (STAT6ko → STAT6ko), and from IL-4/IL-13ko RetnlaCre_R26tdTomato donors into recipients lacking STAT6 (4-13ko → STAT6ko). The upper dot plots show gating strategy for transferred cells, and the lower dot plots indicate percentages of RELMα_tdTomato+ monocyte-derived pMΦs harvested from the peritoneal cavity of recipient mice at day 6 after transfer. Bar diagram summarizes the cell frequencies (mean + SD) from four to five mice per group (pooled from two independent experiments) and points indicate individual mice (see also fig. S8B). *P < 0.05; **P < 0.01 by two-tailed Student’s t test.

  • Fig. 5 RELMα_tdTomato+ Int-MΦs increase whereas Alv-MΦs decline after N. brasiliensis infection.

    (A) WT and STAT6ko RetnlaCre_R26tdTomato mice were infected with N. brasiliensis, and lungs were analyzed at 11 and 20 days p.i. Dot plots show simplified gating of total CD68+ Siglec-F Int-MΦs, CD68+ Siglec-F+ Alv-MΦs (gate R1), and CD68 Siglec-F+ lung eosinophils (gate R2). (B and C) Dot plots show percentages of RELMα_tdTomato+ cells among Int-MΦs and Alv-MΦs (B) or eosinophils (C) in WT versus STAT6ko RetnlaCre_R26tdTomato mice at day 20 p.i. (D and E) Bar diagrams indicate percentages (mean + SD) from total cells (D) or percentages of RELMα_tdTomato-expressing cells (E) among the indicated populations of WT and STAT6ko mice at day 11 (top) and day 20 p.i. (bottom). Note the requirement of STAT6 for induction of the RELMα_tdTomato+ Int-MΦs and eosinophils after infection (E). Data are representative of four independent experiments with at least three to four mice per group. *P < 0.05; **P < 0.01 by two-tailed Student’s t test. ns, not significant. (F) RELMα_tdTomato monocytes of Ly5.2 RetnlaCre_R26tdTomato mice were transferred intravenously into congenic Ly5.1 B6 mice, naïve or infected with N. brasiliensis 3 days before. Lungs of recipient mice were analyzed on day 37 post transfer (p.t.). Representative dot plots of two independent experiments show gating strategy of the donor-derived Ly5.2+ RELMα_tdTomato+ cells, which differentiated into MHC-II+ Siglec-F interstitial and Siglec-F+ MHC-II Alv-MΦs. Bar diagram summarizes the cell frequencies (mean + SD) from four mice per group, and points indicate individual mice (see also fig. S8C). *P < 0.05 by two-tailed Student’s t test.

  • Fig. 6 Requirement of RELMα-expressing cells for prevention of fatal primary and protection during secondary N. brasiliensis infection.

    (A and B) To test the efficiency of DT-mediated depletion of RELMα+ cells, naïve RetnlaCre_R26iDTR mice were either left untreated or injected intraperitoneally with DT at day 0 and day 1, and blood, peritoneal exudates, skin, and lung samples were harvested on day 2 p.i. Representative dot plots (A and fig. S10B) and bar diagrams (B) show percentages + SD of RELMα_tdTomato+ cells among the indicated cell populations from three to four untreated (gray bars) and DT-treated mice (black bars). Data are representative of two independent experiments. *P < 0.05; **P < 0.01 by two-tailed Student’s t test. (C) RetnlaCre+_R26iDTR mice were treated with DT daily from day 0 until day 3 and concomitantly subcutaneously infected with N. brasiliensis (+DT +Nb, n = 7) or left uninfected (+DT, n = 6). Alterations to the well-being of the mice were monitored daily through day 11 after primary infection, and mice exhibiting clinical signs of debilitating phenotype (heavy or slow breathing, severely reduced movement or body weight, and low body temperature) were euthanized and removed from the study. **P = 0.0005 by log-rank test. (D) RetnlaCre+_R26iDTR mice were subcutaneously infected with N. brasiliensis, challenged for the second time with 500 larvae per mouse 30 days after primary infection, and daily treated (+DT) or not (−DT) with DT at days 0 until day 4 during secondary infection. Numbers of retained larvae in the skin at the site of infection (left plot) or numbers of larvae in the lung (middle plot) were analyzed at days 5 and 2 after secondary challenge, respectively. Plot on the right shows analysis of N. brasiliensis worm burden in the small intestine at day 5 after secondary infection. Lines indicate the means, and points indicate individual mice pooled from two independent experiments. *P < 0.05; **P < 0.01 by two-tailed Student’s t test. (E) Image on the right shows severe lung damage in RetnlaCre+_R26iDTR mice challenged for the second time with N. brasiliensis and treated with DT as in (D). DT injection in infected mice lacking the RetnlaCre transgene (left image), as well as secondary N. brasiliensis infection per se (middle image), caused no significant lung damage.

  • Fig. 7 Depletion of RELMα-expressing MΦs compromises survival during primary and control of lung tissue repair and intestinal parasite burden in the secondary N. brasiliensis infection.

    (A) RetnlaCre_CD115iDTR mice (Cre+, n = 5) and Cre-negative controls (Cre, n = 8) were treated with DT on days 4, 5, and 7 after primary N. brasiliensis infection. Alterations to the well-being of the mice were monitored as in Fig. 6C. **P = 0.0026 by log-rank test. (B to E) Lungs of RetnlaCre_CD115iDTR mice were analyzed at day 5 after secondary N. brasiliensis infection and DT treatment as in Fig. 6D. (B) Dot plots and bar diagram indicate percentages of CD11b+ CD11c+ Int-MΦs from total lung cells in naïve, N. brasiliensis-infected RetnlaCre, and RetnlaCre+_CD115iDTR mice. Bars indicate means + SD of four individual mice per group and are representative of two independent experiments. (C) Image on the right displays aggravated lung damage in RetnlaCre+_CD115iDTR mice versus Cre-negative controls (left image), both challenged for the second time with N. brasiliensis and treated with DT. (D) Expression of genes that characterize AAMs in lungs of DT-treated mice (+DT) compared to untreated controls (−DT). (E) Parasite burden in the indicated organs of RetnlaCre+_CD115iDTR mice (Cre+) and Cre-negative controls (Cre), both treated with DT. Data are representative of two independent experiments. *P < 0.05; **P < 0.01 by two-tailed Student’s t test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaau3814/DC1

    Raw data

    Methods

    Fig. S1. Generation of RetnlaCre mice.

    Fig. S2. Structure and sequence information on the recombined BAC vector and its copy number in the genome.

    Fig. S3. RELMα_tdTomato expression correlates with Retnla mRNA and RELMα protein expression and can be induced in vitro by IL-4.

    Fig. S4. Immunofluorescence analysis of RELMα_tdTomato expression in selected organs of RetnlaCre_R26tdTomato mice.

    Fig. S5. Ex vivo RELMα_tdTomato fluorescence imaging of selected organs of naïve RetnlaCre_R26tdTomato mice.

    Fig. S6. Flow cytometry gating strategies for the indicated cell types isolated from naïve RetnlaCre_R26tdTomato mice.

    Fig. S7. Analysis of RELMα_tdTomato-expressing Alv-MΦs in RetnlaCre_R26tdTomato mice.

    Fig. S8. RELMα_tdTomato expression in MΦs derived from adoptively transferred monocytes.

    Fig. S9. Analysis of MΦs in the lungs of RetnlaCre_R26tdTomato mice infected with N. brasiliensis.

    Fig. S10. Depletion efficiency of RELMα_tdTomato+ cells after DT treatment of RetnlaCre_R26tdTomato/iDTR mice.

    Table S1. List of antibodies used for flow cytometry and immunofluorescence.

    Table S2. Sequences of primers used for quantitative RT-PCR.

    References (4245)

  • Supplementary Materials

    The PDF file includes:

    • Raw data
    • Methods
    • Fig. S1. Generation of RetnlaCre mice.
    • Fig. S2. Structure and sequence information on the recombined BAC vector and its copy number in the genome.
    • Fig. S3. RELMα_tdTomato expression correlates with Retnla mRNA and RELMα protein expression and can be induced in vitro by IL-4.
    • Fig. S4. Immunofluorescence analysis of RELMα_tdTomato expression in selected organs of RetnlaCre_R26tdTomato mice.
    • Fig. S5. Ex vivo RELMα_tdTomato fluorescence imaging of selected organs of naïve RetnlaCre_R26tdTomato mice.
    • Fig. S6. Flow cytometry gating strategies for the indicated cell types isolated from naïve RetnlaCre_R26tdTomato mice.
    • Fig. S7. Analysis of RELMα_tdTomato-expressing Alv-MΦs in RetnlaCre_R26tdTomato mice.
    • Fig. S8. RELMα_tdTomato expression in MΦs derived from adoptively transferred monocytes.
    • Fig. S9. Analysis of MΦs in the lungs of RetnlaCre_R26tdTomato mice infected with N. brasiliensis.
    • Fig. S10. Depletion efficiency of RELMα_tdTomato+ cells after DT treatment of RetnlaCre_R26tdTomato/iDTR mice.
    • Table S1. List of antibodies used for flow cytometry and immunofluorescence.
    • Table S2. Sequences of primers used for quantitative RT-PCR.
    • References (4245)

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